Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis and decreased blood counts. We have shown that the p38 MARK pathway is constitutively activated in MDS bone marrows and plays a role in the increased apoptosis seen in hematopoietic progenitors. Most importantly, pharmacological inhibition of p38 leads to increased hematopoietic colony formation from primary MDS CD34+ hematopoietic progenitors and leads to enhanced hematopoiesis in a variety of MDS subtypes in vitro. This proposal will define the pathophysiological role that p38 plays in MDS, and will identify its molecular and cellular targets. Specific Aim 1 will determine whether constitutive p38 activation results in ineffective hematopoiesis in MDS. We will selectively inhibit the expression of the various p38 isoforms (alpha, beta, gamma, delta) in CD34+ cells from MDS bone marrows and examine the effects of such inhibition on hematopoietic progenitor colony formation and apoptosis. The effects of adenoviral-mediated overexpression of various isoform-specific dominant negative mutants on hematopoietic progenitor cell growth will also be assessed. The effects seen will be correlated with various MDS subtypes and clinical characteristics. Specific Aim 2 will study the mechanisms of constitutive activation of p38 in MDS and identify its downstream effectors. Biochemical, immunohistochemical and flow cytometric methodologies will be used to examine the activation of putative upstream and downstream effectors in bone marrow-derived hematopoietic progenitors. These will include evaluation of the activation status of the small G-protein Rac1 and the upstream Map kinase kinases, MKK3, MKK6 and MKK4, as well as the activation of the downstream effectors MapKapK-2, MapKapK-3, and Msk1. siRNA-mediated knockdown of kinases found constitutively activated will be subsequently used to determine the functional relevance of each one of them in MDS hematopoiesis. Bone marrow microenvironment can also contribute to the pathophysiology of MDS by being involved in cytokine secretion. Thus, Specific Aim 3 is to examine whether activation of p38 MARK mediates overproduction of myelosuppressive cytokines in MDS bone marrows. We will examine whether p38 inhibitors suppress the overproduction of TNFalpha and IFNgamma by infiltrating macrophages and lymphocytes in the bone marrows of MDS patients. We will also evaluate IL-6 and VEGF production by MDS marrow derived stromal cells as compared to normal stromal cells and assess the effects of p38 inhibitors on such production. Altogether, these studies should provide valuable information on the role of p38 MARK pathway in the pathogenesis of MDS. Moreover, the results of these studies should be of direct clinical-translational relevance and lead to the development of novel therapeutic approaches for the treatment of MDS, including clinical trials with clinically relevant pharmacological inhibitors of p38 pathway such as SCIO-469.